The ubiquitous second messenger, cAMP, is an important modulator for the development of the immune system and for various immune responses. Its effects range from development of hematopoietic lineages to B cell and T cell activation, cytokine generation, and chemotaxis. To ensure the proper function of this second messenger it is regulated by numerous signaling pathways at multiple levels. Intracellular cAMP is synthesized by membrane-bound adenylyl cyclases (ACs) upon activation of the heterotrimeric G1s protein. There are nine membrane-bound ACs, each of which is distinctively regulated by a variety of pathways including Gi, G23, Ca2+, and protein kinase C. Thus the ACs can serve as key integrators for inputs from multiple pathways. Despite the crucial functions of cAMP in the immune system, its regulation is not well understood. We have recently discovered a novel pathway that regulates cAMP responses in several cell lines including primary macrophages derived from bone marrows. In these cells, activation of the G113 pathway by distinct G protein coupled receptors synergistically enhances Gs-activated synthesis of cAMP. This synergistic interaction converges on a specific isoform of adenylyl cyclase (AC), AC7, which is highly expressed in the immune system. This is the first demonstration that the G12/G13 subclass of G proteins can regulate cAMP responses and of a specific function for AC7. We also found that deficiency of AC7 in the immune system results in compromised immune responses, suggesting an important role of this AC isoform in the immune functions. We propose to determine the molecular mechanisms of AC7 regulation and to elucidate its physiological functions in the immune system. Three specific aims are proposed to achieve these goals. 1. We will dissect the molecular mechanisms of the interactions between the G12/13 pathway and AC7 using a variety of biochemical approaches. We will determine the requirements for this synergistic interaction to occur and the mechanism that turns off this transient interaction. 2. We will identify novel proteins involved in the regulation of AC7 activity by the G12/13 pathway using a combination of protein-protein interaction assays and an RNAi screen. 3. We will identify the defects in chimeric mice with AC7 deficient immune systems, define the causes of the defects in specific cell types, and ascertain the signaling pathways involved. We will also generate a conditional knockout strain of AC7 to use it for more advanced studies of AC7 functions in immune responses and eventual elucidation of the role of this cyclase in other tissues and cell types. Results from this study will help us better understand the regulation of cAMP and its physiological role in the immune system. Knowledge gained through elucidation of these novel pathways and regulatory paradigms will help identify selective drug targets for combating immune diseases. PUBLIC HEALTH RELEVANCE: Multiple drugs have been developed based on their abilities to modulate cAMP and they have successfully alleviated symptoms of several abnormal conditions, including selected cardiac conditions, pulmonary dysfunction, addiction, depression, and immune functions. This application will elucidate the mechanisms for a novel regulation of cAMP in immune cells and determine how this regulation affects immune functions in mice. Results from these studies will help identify novel targets for selective therapeutic intervention in immune diseases.